Regulation of osteopontin

ABSTRACT

The present invention provides methods for the regulation of osteopontin activity in a subject as well as for treating or preventing conditions associated with an increased activity of osteopontin activity in a subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to, and is a continuation of U.S.application Ser. No. 12/045,623, filed Mar. 10, 2008, which claimspriority to Provisional Application Ser. No. 60/894,153, entitled“Regulation of Osteopontin,” filed Mar. 9, 2007, each of which isincorporated herein by reference in their entirety.

Osteopontin (“OPN”), also known as secreted phosphoprotein 1 (“SPP1”),early T-lymphocyte activation marker (“Eta-1”), sialoprotein 1 or 44KBPP (bone phosphoprotein), is a glycosylated phosphoprotein found inplasma, other bodily fluids, and extracellular matrices. The protein iscomposed of approximately 300 amino acids residues and has about 30carbohydrate residues, including 10 sialic acid residues, attached toit. OPN is an acidic protein which exhibits a high amino acid homologybetween species (e.g., mouse, rat, human and pig) with several conservedelements including a stretch of 7 to 9 Asp or Glu residues.

Osteopontin is biosynthesized by a variety of tissue types includingpreosteoblasts, osteoblasts, osteocytes, extraosseous cells in the innerear, brain, kidney, deciduum, placenta, odontoblasts, some bone marrowcells, hypertrophic chondrocytes, macrophages, smooth muscle, andendothelial cells. In the bone, the protein is primarily made by cellsof the osteoblastic lineage and deposited on mineralized matrix. It isabundant in bone mineral matrix and accelerates bone regeneration andremodeling. Osteopontin is a multifunctional protein with an ability tobind several proteins, including integrin proteins and variants of theprotein CD44.

Osteopontin is associated with, and plays a role in, the regulation andprogression of many diseases. Osteopontin is known to be increased in anumber of autoimmune disorders and is overexpressed in a variety ofcancers. Plasma levels of osteopontin are also elevated in individualswith coronary artery disease and elevated levels of osteopontin arefound in the synovial fluid of individuals with rheumatoid arthritis.

Modulation of osteopontin may, therefore, confer significant therapeuticbenefits. Accordingly, there is a need to identify means to regulateosteopontin, especially means to lower the effective osteopontin levelor concentration.

The present invention is based, at least in part, on the discovery thatosteopontin can be regulated by regulating S-adenosyl methioninedecarboxylase (AMD1), polyamine biosynthesis, adenosine or a pathwaycontaining either AMD1 or adenosine. Accordingly, the present inventionprovides methods for the regulation of osteopontin activity in a subjectas well as for treating or preventing conditions associated with anincreased activity of osteopontin activity in a subject.

The present invention provides a method of decreasing the activity ofosteopontin in a cell. The method comprises contacting a cell with aneffective amount of an agent that inhibits S-adenosyl methioninedecarboxylase (“AMD1”), inhibits polyamine biosynthesis, or increasesadenosine in the cell.

According to another aspect, the present invention provides a method ofdecreasing the activity of osteopontin in a cell by contacting the cellwith an effective amount of MGBG, a salt of MGBG, or a protectedderivative of MGBG.

According to another aspect, the present invention provides a method oftreating or preventing a condition associated with an increased activityof osteopontin. The method comprises administering to a subject in needof such treatment an effective amount of an agent that inhibitsS-adenosyl methionine decarboxylase, inhibits polyamine biosynthesis, orincreases adenosine in the subject, with the proviso that the agent isnot MGBG, a polyamine analog or a salt or protected derivative thereof.

According to yet another aspect, the present invention provides a methodof treating a condition. The method comprises administering to a subjectin need of such treatment an effective amount of MGBG, a salt of MGBG, aprotected derivative of MGBG, or a polyamine analog, a salt, a protectedderivative, or a stereoisomer thereof, wherein the condition is selectedfrom the group consisting of Crohn's disease, Parkinson's disease,inflammatory bowel disorder, multiple sclerosis (MS), amyotrophiclateral sclerosis (ALS), hepatitis, HBV, HCV, nephritis, cerebritis,glomerulonephritis, rheumatoid arthritis, type 2 diabetes, cardiacfibrosis and angiotensin type II associated hypertension, osteoporosis,a mast cell produced IgE mediated hypersensitivity immune reaction,peripheral sensory neuropathy associated with HIV infection or diabetesmellitus, asthma, autism, dermatomyositis, frailty, obesity, primarybiliary cirrhosis, primary sclerosing cholangitis, post-radiationsyndrome, psoriatic arthritis, sarcoidosis, scleroderma with or withoutpulmonary fibrosis, a kidney related autoimmune condition, diabeticnephropathy, a diabetic vascular complication, and a lymphoproliferationrelated autoimmune condition.

According to yet another aspect, the present invention provides a methodof decreasing osteopontin secretion from monocytes or macrophages. Themethod comprises contacting a monocyte or macrophage with an effectiveamount of an agent that inhibits S-adenosyl methionine decarboxylase,inhibits polyamine biosynthesis, or increases adenosine in the monocyteor macrophage.

According to yet another aspect, the present invention provides a methodof decreasing osteopontin secretion from monocytes or macrophages. Themethod comprises contacting a monocyte or macrophage with an effectiveamount of an agent that inhibits S-adenosyl methionine decarboxylase,inhibits polyamine biosynthesis, or increases adenosine in the monocyteor macrophage.

According to yet another aspect, the present invention provides a methodof decreasing differentiation of macrophages from monocytes. The methodcomprises contacting a monocyte with an effective amount of an agentthat inhibits S-adenosyl methionine decarboxylase, inhibits polyaminebiosynthesis, or increases adenosine in the monocyte.

These and other features of the present invention are set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the drawings are forillustration purposes only. The drawings are not intended to limit thescope of the present invention in any way.

FIG. 1 shows the structures of MGBG, SL47 (also referred to as SL11047)and SL 93 (also referred to as SL11093).

FIG. 2 depicts the changes in cell counts and RNA signals of OPN, ADAand other genes induced by MGBG in mononuclear cells separated on thebasis of cell-surface CD14 and CD16 expression.

FIG. 3 depicts the average OPN levels in CCS of breast cancer and normalPBMCs treated with MGBG.

FIG. 4 depicts the average OPN levels in CCS of AD and normal PBMCstreated with MGBG.

FIG. 5 depicts the average OPN levels in CCS of HIV and normal PBMCstreated with MGBG.

FIG. 6 depicts the average OPN levels in CCS of ALS and normal PBMCstreated with MGBG.

FIG. 7 depicts the average OPN levels in CCS of AIDS dementia and normalPBMCs treated with MGBG.

FIG. 8 shows that rOPN increases CD14+/CD16+ cell population.

FIG. 9 shows the results with, and without MGBG (PA001) treatment ininfected macaques on macrophages in CNS.

FIG. 10 shows the OPN levels in brains of SIV-infected animals with, andwithout, MGBG treatment.

FIG. 11 shows the OPN levels in lymph nodes of SIV-infected animalswith, and without, MGBG treatment. Samples B146 and CB18 were treatedwith MGBG, whereas BT66 was not.

FIG. 12 shows the effect of MGBG on Osteopontin RNA in PBMC cultures.

FIG. 13 shows the effect of MGBG on Osteopontin Protein Production inPBMC cultures.

The present invention is based, at least in part, on the discovery thatosteopontin can be regulated by regulating S-adenosyl methioninedecarboxylase (“AMD1”), polyamine biosynthesis, adenosine or a pathwaycontaining either AMD1 or adenosine. Accordingly, the present inventionprovides methods for the regulation of osteopontin activity in a subjectas well as for treating or preventing conditions associated with anincreased activity of osteopontin activity in a subject.

According to one aspect, the present invention provides a method ofdecreasing the activity of osteopontin in a cell. The term “osteopontin”is used interchangeably with “OPN,” “SPP1,” “Eta-1,” sialoprotein 1 or44K BPP (bone phosphoprotein). In general, osteopontin refers to anyfull-length or partial fragment of a full-length osteopontin.Osteopontin can also refer to any modified, e.g., glycosylated,osteopontin.

The term “activity” as used herein refers to both, the biologicalactivity of the polypeptide and to the quantity or level of osteopontinpresent in the cell. In one embodiment, the term activity refers to thequantity of osteopontin, e.g., present, expressed or produced in thecell. In another embodiment, it refers to the level of osteopontinsecreted by the cell, for example, by a mononuclear cell.

According to another aspect, the present invention provides a method oftreating or preventing a condition associated with an increased activityof osteopontin. The method comprises administering to a subject in needof such treatment an effective amount of an agent that regulates theactivity of osteopontin. The condition can be any condition now known,or later discovered, to be associated with an increased activity ofosteopontin. Examples of conditions associated with an increasedactivity of osteopontin include, but are not limited to, autoimmunediseases, inflammatory diseases, neoplastic growth and tumor metastases.In one embodiment, the condition associated with an increased activityof osteopontin is infiltration of immune cells to an affected area orincreased level of CD14/CD16 macrophages in a subject.

In another embodiment, conditions associated with an increased activityof osteopontin include, but are not limited to, multiple sclerosis (MS),atherosclerosis and related coronary diseases, rheumatoid arthritis,lupus, nephritis, cerebritis, Crohn's disease, osteoporosis,inflammatory bowel disorder, breast cancer, ovarian cancer, pancreaticcancer, bladder cancer, lung cancer, colon cancer, gastric carcinomas,esophageal carcinomas, squamous cell carcinomas of the head or neck,prostate cancer, thyroid cancer, melanoma, kidney cancers, renal cellcarcinomas, endometrial cancer, small intestine cancer, duodenal cancer,cholangiocarcinoma, astrocytoma, AIDS lymphoma, follicular lymphoma,T-cell lymphoma, B-cell lymphoma, proliferative retinopathy,vitreoretinopathy, diabetic retinopathy, macular degeneration, non-HIVdementia, HIV- and AIDS-associated dementia, focal segmentalglomerulosclerosis, membrane proliferative glomerulonephropathy,psoriasis, herpes virus associated disease, Castleman's disease,Kaposi's sarcoma, Alzheimer's disease, type 2 diabetes, cardiac fibrosisand angiotensin type II associated hypertension, mast cell produced IgEmediated hypersensitivity immune reactions, prelymphomatic orlymphoproliferation related autoimmune conditions, angioimmunoblasticlymphadenophathy (AILD), glomerulonephritis and other glomerulardiseases, immunoglobulin A (IgA) nephropathy, Amyotrophic LateralSclerosis (ALS), hepatitis including HBV and HCV, peripheral sensoryneuropathy associated with HIV infection or diabetes mellitus, asthma,autism, dermatomyositis, frailty, obesity, Parkinson's disease, primarybiliary cirrhosis, primary sclerosing cholangitis, post-radiationsyndrome, psoriatic arthritis, sarcoidosis, scleroderma with or withoutpulmonary fibrosis, kidney related autoimmune conditions, diabeticnephropathy and other diabetic vascular complications.

In yet another embodiment, the condition associated with an increasedactivity of osteopontin is not associated with macrophage proliferation.

A “subject” may be any animal suffering from a condition associated withan increased activity of osteopontin that is treatable in accordancewith the methods of the invention. An animal is a living multicellularvertebrate organism, and includes both human and non-human mammals.

An “effective amount” or a “therapeutically effective amount” is aquantity of a compound (e.g., MGBG, a polyamine analog or any agent)that is sufficient to achieve a desired effect in a subject beingtreated. For instance, this can be the amount necessary to reduceosteopontin activity or to otherwise measurably alter or alleviate thesymptoms of increased osteopontin activity. The effective amount of acompound of the present invention may vary depending upon the route ofadministration and dosage form. In addition, specific dosages may beadjusted depending on conditions of disease, the age, body weight,general health conditions, sex, and diet of the subject, dose intervals,administration routes, excretion rate, and combinations of agents.

According to yet another aspect, the present invention provides a methodof decreasing osteopontin secretion from monocytes or macrophages. Themethod comprises contacting a monocyte or macrophage with an effectiveamount of an agent that regulates the activity of osteopontin.

According to yet another aspect, the present invention provides a methodof decreasing differentiation of macrophages from monocytes. The methodcomprises contacting a monocyte with an effective amount of an agentthat regulates the activity of osteopontin.

The agent useful in the methods of the invention can be any agent thatdecreases the activity of osteopontin. In one embodiment, the agent iscapable of inhibiting S-adenosyl methionine decarboxylase (“AMD1”) orany pathway containing AMD1, e.g., any entity upstream or downstream ofa pathway containing AMD1, especially any pathway containing AMD1 andassociated with adenosine production. In another embodiment the agent iscapable of inhibiting polyamine biosynthesis or any pathway involved inpolyamine biosynthesis.

Alternatively the agent is capable of increasing the activity ofadenosine in the cell, either directly, or via a pathway containingadenosine. In general, a pathway containing AMD1 or adenosine isunderstood to refer to a pathway in which either AMD1 or adenosine isinvolved, including, for example, as a substrate, catalyst, product orby-product.

The agent can be any kind of known or later discovered agent that caninhibit the activity of the enzyme S-adenosyl methionine decarboxylase,can inhibit polyamine biosynthesis, or that can increase the activity ofadenosine in, for example, a cell. In one embodiment, the agent is achemical agent, including, but not limited to, organic molecules andsalts, protected derivatives and stereoisomers thereof, inorganicmolecules or various ionic or elemental entities. In another embodiment,the agent is a biological agent or a biomolecule, for example, apolypeptide, an antibody or an active fragment thereof, or a nucleicacid molecule, e.g., RNAi.

According to one embodiment of the present invention, the agent is apolyamine analog or a salt, a protected derivative, or a stereoisomerthereof. Any polyamine analog is suitable for use in the methods of thepresent invention. Exemplary polyamine analogs used in the methods ofthe invention include compounds of the structures 1, 2, 3, 4, and 5, andthe corresponding stereoisomers, salts, and protected derivativesthereof:

where R₁, R₂, R₄, R₆ and R₇ are independently selected from the groupconsisting of hydrogen, alkyl and aryl, and where R₃ and R₅ are alkylgroups;

where R1, R2, R4, R6, R8, and R9 are independently selected from thegroup consisting of hydrogen, alkyl and aryl and where R3, R5 and R7 arealkyl groups;

where R₁, R₂, R₄, R₆, R₁₀ and R₁₁ are independently selected from thegroup consisting of hydrogen, alkyl and aryl, and where R₃, R₅, R₇ andR₉ are alkyl groups;

where R₁ and R₅ are independently selected from the group consisting ofmethyl, ethyl, n-propyl, and isopropyl;where R₂, R₃, and R₄ are independently selected from the groupconsisting of C₁-C₆ alkyl, C₂-C₆ alkenyl, C₃-C₆ cycloalkyl, C₁-C₆alkyl-C₃-C₆ cycloalkyl-C₁-C₆ alkyl, C₃-C₁₀ aryl, and C₁-C₆ alkyl-C₃-C₁₀aryl-C₁-C₆ alkyl;and where R₆, R₇, R₈ and R₉ are independently selected from the groupconsisting of H, methyl, and ethyl;

where R₁ and R₆ are independently selected from the group consisting ofmethyl, ethyl, n-propyl, and isopropyl;R₂, R₃, R₄ and R₅ are independently selected from the group consistingof C₁-C₆ alkyl, C₂-C₆ alkenyl, C₃-C₆ cycloalkyl, C₁-C₆ alkyl-C₃-C₆cycloalkyl-C₁-C₆ alkyl, C₃-C₁₀ aryl, and C₁-C₆ aryl-C₁-C₆ alkyl;and where R₇, R₈, R₉, R₁₀ and R₁₁ are independently selected from thegroup consisting of H, methyl, and ethyl.

In another embodiment, the polyamine analogs are compounds of thestructures 2 and 3, where R₃, R₅, R₇ and R₉ are independently (CH₂)_(x)groups, where x is an integer from 2 to 6, and further where R₄, R₆ andR₈ are hydrogen atoms.

In yet another embodiment, the polyamine analogs are compounds of thestructures 2 and 3, where R₃, R₅, R₇ and R₉ are independently (CH₂)₂groups, where x is an integer from 2 to 6, and where R₄, R₆ and R₈ arehydrogen atoms, and where R₁ and R₁₀ are alkyl groups, and further whereR₂ and R₁₁ are hydrogen atoms.

In yet another embodiment, the polyamine analogs are compounds of thestructures 2 and 3, where R₃, R₅, R₇ and R₉ are independently (CH₂)₂groups, where x is an integer from 2 to 6, and where R₄, R₆ and R₈ arehydrogen atoms, and where R₁ and R₁₀ are alkyl groups, and where R₂ andR₁₁ are hydrogen atoms, and further where the polyamine analogs have amolecular weight less than 500.

Further embodiments of compounds of the structure 4 include those whereR₆, R₇, R₈ and R₉ are H;

where R₁ and R₅ are ethyl;where R₆, R₇, R₈ and R₉ are H and R₁ and R₅ are ethyl;and/or where R₂ and R₄ are independently selected from the groupconsisting of C₁-C₆ alkyl andR₃ is selected from the group consisting of C₁-C₆ alkyl, C₂-C₆ alkenyl,C₃-C₆ cycloalkyl, C₁-C₆ alkyl-C₃-C₆ cycloalkyl-C₁-C₆ alkyl, C₃-C₁₀ aryl,and C₁-C₆ alkyl-C₃-C₁₀ aryl-C₁-C₆ alkyl.

Additional polyamine analogs useful in the present invention includecompounds of the formula 6, and the corresponding stereoisomers, salts,and protected derivatives thereof:

where R₄ is C₂-C₆ n-alkenyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloalkenyl, orC₃-C₆ aryl;R₃ and R₅ are independently chosen from a single bond, C₁-C₆ alkyl, orC₁-C₆ alkenyl;R₂ and R₆ are independently chosen from C₁-C₆ alkyl, C₁-C₆ alkenyl,C₃-C₆ cycloalkyl, C₃-C₆ cycloalkenyl, or C₃-C₆ aryl;R₁ and R₇ are independently chosen from H, C₁-C₆ alkyl, or C₂-C₆alkenyl; and

R₈, R₉, R₁₀, and R₁₁ are H.

In certain embodiments of the compounds of formula 6, R₁ and R₇ areindependently chosen from C₁-C₆ alkyl or C₂-C₆ alkenyl.

Additional polyamine analogs useful in the present invention includecompounds of the formula 7, and the corresponding stereoisomers, salts,and protected derivatives thereof:

where R₄ is C₁-C₆ n-alkyl or C₁-C₆ branched alkyl;R₃ and R₅ are independently chosen from a single bond or C₁-C₆ alkyl;R₂ and R₆ are independently chosen from C₁-C₆ alkyl, C₁-C₆ alkenyl,C₃-C₆ cycloalkyl, C₃-C₆ cycloalkenyl, or C₃-C₆ aryl;R₁ and R₇ are independently chosen from H, C₁-C₆ alkyl, or C₂-C₆alkenyl; and

R₈, R₉, R₁₀, and R₁₁ are H.

In certain embodiments of the compounds of formula 7, R₂ and R₇ areindependently chosen from C₁-C₆ alkyl or C₂-C₆ alkenyl, R₄ is C₁-C₆saturated n-alkyl or C₁-C₆ saturated branched alkyl, and R₃ and R₅ areindependently chosen from a single bond or C₁-C₆ saturated n-alkyl.

As used herein, a “polyamine” is any of a group of aliphatic,straight-chain amines derived biosynthetically from amino acids;polyamines are reviewed in Marton et al. (1995) Ann. Rev. Pharm.Toxicol. 35:55-91. By “polyamine” is generally meant anaturally-occurring polyamine or a polyamine which is naturally producedin eukaryotic cells. Examples of polyamines include putrescine,spermidine, spermine and cadaverine.

As used herein, a “polyamine analog” is an organic cation structurallysimilar but non-identical to naturally-occurring polyamines such asspermine and/or spermidine and their precursor, diamine putrescine.Polyamine analogs can be branched or un-branched, or incorporate cyclicmoieties. Polyamines may comprise primary, secondary, tertiary, orquaternary amino groups. In one embodiment, all the nitrogen atoms ofthe polyamine analogs are independently secondary, tertiary, orquaternary amino groups, but are not so limited. Polyamine analogs mayinclude imine, amidine and guanidine groups in place of amine groups.The term “polyamine analog” includes stereoisomers, salts and protectedderivatives of polyamine analogs.

A “stereoisomer” is any optical isomer of a compound, includingenantiomers and diastereomers. Unless otherwise indicated, structuralformulae of compounds are intended to embrace all possiblestereoisomers.

A “salt” or “pharmaceutically acceptable salt” is a compound formed bythe replacement of one or more hydrogen atoms with elements or groups,which is composed of anions and cations, which usually ionizes in water;a salt is formed, for instance, by neutralization of an acid by a base.Examples of salts include, but are not limited to, halide, for example,chloride, bromide, or iodide, nitrate, sulfate, bisulfate, phosphate,acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate,tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate,gentisinate, fumarate, gluconate, glucaronate, saccharate, formate,benzoate, glutamate, methanesulfonate, ethanesulfonate, benzensulfonate,p-toluenesulfonate and pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts.

“Protected derivative” is used to refer to a compound protected with aprotecting group. “Protecting group” refers to a chemical group thatexhibits the following characteristics: 1) reacts selectively with thedesired functionality in good yield (preferably at least 80%, morepreferably at least 90%, more preferably at least 95%, still morepreferably at least 99%) to give a protected substrate that is stable tothe projected reactions for which protection is desired; 2) isselectively removable from the protected substrate to yield the desiredfunctionality; and 3) is removable in good yield (preferably at least80%, more preferably at least 90%, more preferably at least 95%, stillmore preferably at least 99%) by reagents compatible with the otherfunctional group(s) present or generated in such projected reactions.Examples of suitable protecting groups can be found in Greene et al.(1991) Protective Groups in Organic Synthesis, 2nd Ed. (John Wiley &Sons, Inc., New York). Exemplary protecting groups for the aminofunctionality include, but are not limited to, mesitylenesulfonyl(MesSO₂), benzyloxycarbonyl (CBz), t-butyloxycarbonyl (Boc),t-butyldimethylsilyl (TBDIMS), 9-fluorenylmethyloxycarbonyl (Fmoc), orsuitable photolabile protecting groups such as 6-nitroveratryloxycarbonyl (Nvoc).

An “alkyl” is a cyclic, branched, or straight chain chemical groupcontaining carbon and hydrogen, such as methyl, butyl, t-butyl, pentyl,cyclopropyl, and octyl. Alkyl groups can be either unsubstituted orsubstituted with one or more substituents, e.g., halogen, alkoxy,acyloxy, amino, hydroxyl, mercapto, carboxy, benzyl. Alkyl groups can besaturated or unsaturated (e.g., containing —C═C— or ˜C≡C— subunits), atone or several positions. Unless otherwise specified, alkyl groups willcomprise 1 to 8 carbon atoms, but may include 1 to 6, or even 1 to 4carbon atoms. “Cycloalkyl” refers to cyclic alkyl groups only, such ascyclopropyl, cyclobutyl, cyclopentyl, etc. “n-alkyl” refers to a linear(i.e., straight-chain) alkyl group only, while “branched alkyl” refersto branched alkyl groups to the exclusion of cyclic and linear alkylgroups. “Alkenyl” refers to a cyclic, branched, or straight chainchemical group containing carbon and hydrogen where at least one bond ismonounsaturated, such as ethenyl, cyclopentenyl, or 1,3-butadienyl.Alkenyl groups can be substituted as indicated for alkyl groups. Alkenylgroups can be designated as cyclic, linear (n-alkenyl) or branched in ananalogous fashion to the preceding designations for alkyl. An “aryl” isan unsaturated aromatic carbocyclic group having a single ring (e.g.,phenyl), or multiple condensed rings (e.g., naphthyl), which canoptionally be unsubstituted or substituted with amino, hydroxyl, alkyl,alkoxy, chloro, halo, mercapto and other substituents.

According to another embodiment of the present invention, the agent is achemical moiety that inhibits the activity of S-adenosyl methioninedecarboxylase, inhibits polyamine biosynthesis, and/or increases theactivity of adenosine. Examples of such moieties include, but are notlimited to, those listed in Table 1. Irrespective of the form of themoiety listed in Table 1, it is understood that it includes, asapplicable, a salt, protected derivative, and stereoisomer thereof.

TABLE 1 Exemplary Inhibitors of S-adenosyl methionine decarboxylaseand/or polyamine biosynthesis Pub Chem Compound Official Name (NotIUPAC) ID Decarboxylated SAM s-adenosyl-3-methylthiopropylamineMitoguazone or Methylglyoxal bis(guanyl-hydrazone) 5351154 “MGBG” EGBGEthylglyoxal bis(guanylhydrazone) 9561662 Berenil Diminazene orDiminazene aceturate 2354 Pentamidine 4-[5-(4-carbamimidoylphenoxy) 4735pentoxy]benzenecarboximidamide 5′-(Dimethylsulphino)-5′-deoxyadenosineS-adneosyl-4-methylthiobutyrate S-adenosyl-S-methyl-L-cysteine AMAS-(5′-Deoxy-5′-adenosyl) methylthioethylhydroxylamine EMGBGEthylmethylglyoxal bis(guanylhydrazone) 9574151 DEGBG Diethylglyoxalbis(guanylhydrazone) 5479208 CGP-33′8296-((2-carbamimidoylhydrazono)methyl)picolinimidamide CGP-36′958CGP-39′937 2,2′-bipyridine-6,6′-bis(carboximidamide) CGP-48664 or CGP-4-amidinoindan-1-one 2′-amidinohydrazone 5486811 48664A or SAM 364AAbeAdo or MDL-73811 5′-[[(Z)-4-amino-2-butenyl]methylamino]-5′-deoxyadenosine 6436013 MAOEA5′-deoxy-5′-[N-methyl-N-[2-(aminooxy)ethyl]amino]adenosine 3081018 MHZPA5′-deoxy-5′-[N-methyl-N-(3-hydrazinopropyl)amino]adenosine 122092 MHZEA5′-deoxy-5′-[(2-hydrazinoethyl)-methylamino]adenosine AdoMacS-(5′-deoxy-5′-adenosyl)-1-ammonio-4-(methylsulfonio)-2- 3083364cyclopentene AdoMaoS-(5′-deoxy-5′-adenosyl)-1-aminoxy-4-(methylsulfonio)-2-cyclopentene APA1-Aminooxy-3-aminopropane 65020 AOE-PUN-[2-aminooxyethyl]-1,4-diaminobutane AP-APA1-aminooxy-3-N-[3-aminopropyl]-aminopropane 1,11-bis(ethyl)norspermineBES 1,8-bis(ethyl)spermidine BES 1,12-bis(ethyl)spermine DESPMN1,N12-diethylspermine BE-3-3-3 1,11-bis(ethylarnino)-4,8-diazaundecanBE-4-4-4 1,14-bis(ethylamino)-5,10-diazatetradecane DEHOP or DEHSPMDiethylhomospermine, N1,N14-diethylhomospermine DENOPdiethyl-norspermine BE-4-4-4-4 1,19-bis(ethylamino)-5,10,15-triaza-nonadecane SL11037N-ethyl-N′-(2-(3′-ethylamino-propylamino methyl)-cis-cyclopropylmethyl)-propane 1,3-diamine tetrahydrochloride SL11038N-ethyl-N′-(2-(3′-ethylamino-propylamino methyl)-trans-cyclobutylmethyl)-propane 1,3-diamine tetrahydrochloride SL11044N-ethyl-N′-(2-(3′-ethylamino-propylamino methyl)-trans-cyclopropylmethyl)-propane 1,3-diamine tetrahydrochloride SL11047 orSL47 N,N′-bis(3-ethylaminopropyl)-cis-but-2-ene-1,4-diaminetetrahydrochloride SL11093 or SL93N,N′-(cyclopropane-1,2-diylbis(methylene))bis(N4-ethylbutane-1,4-diamine)

In yet another embodiment, the agent is a compound selected from thegroup consisting of MGBG, MDL73811, CGP48664, Berenil, Pentamidine,SL47, and SL93, or a combination of two or more thereof. In yet anotherembodiment, the agent is MGBG, SL47 or SL93. Structures for MGBG, SL47and SL93 are shown in FIG. 1. In still another embodiment, two or moreagents are used in the methods of the invention to regulate the activityof osteopontin. The two or more agents can be used either sequentiallyor simultaneously.

In one embodiment, the agent is a compound selected from the list ofagents listed in Table 1, with the proviso that the agent is not MGBG.In another embodiment, the agent is not a polyamine analog. In yetanother embodiment, the agent is not MGBG or a polyamine analog. In yetanother embodiment, the agent is not a polyamine biosynthesis inhibitor.In yet another embodiment, the agent is a compound selected from thegroup consisting of MDL73811, CGP48664, Berenil and Pentamidine.

“MGBG” is 1,1′ [methylethanediylidene]dinitrilodiguanidine and is alsoknown as methylglyoxal bis(guanylhydrazone), methyl-GAG, andmitoguazone. As used herein, MGBG includes the free base and saltsthereof. It is commonly, but not necessarily, used as a dihydrochloride.

The agent may also be administered in combination with one or moreentities. In one embodiment, the entity is a therapeutic entity,including, but not limited to, a steroid or other anti-inflammatoryagent. In another embodiment, the entity is a pharmaceuticallyacceptable carrier.

The effective amount of an agent that inhibits S-adenosyl methioninedecarboxylase or increases the activity of adenosine, e.g., in a cell ora subject, can be any amount that is sufficient to decrease the activityof osteopontin, e.g., in the cell or the subject, typically by about25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more. In oneembodiment, the effective amount of an agent is an amount that issufficient to decrease the activity of osteopontin by 70% or more. Inanother embodiment, the effective amount of an agent is an amount thatis sufficient to decrease the activity of osteopontin by 80% or more. Inyet another embodiment, the agent inhibits S-adenosyl methioninedecarboxylase and the effective amount is an amount sufficient toactivate adenosine deaminase (“ADA”). In still another embodiment, theagent inhibits S-adenosyl methionine decarboxylase and the effectiveamount is an amount sufficient to increase the activity of adenosine.

The optimal dose, frequency of administration, and duration of treatmentwith the agent that decreases the activity of osteopontin in a subjectmay vary from subject to subject, depending on the subject's condition,the subject's age, weight, response to the treatment, and the nature ofthe therapeutic entity. The optimal dose and duration of treatment maybe best determined by monitoring the subject's response during thecourse of the treatment. In some instances, the administration of higherdoses may permit less frequent administration, and lower doses mayrequire more frequent administration in order to achieve a clinicallysignificant improvement in the subject's condition. The agent may beadministered as a single dose or in multiple doses.

Generally, a therapeutically effective dose of the agent in accordancewith the present methods will be one or more doses of from about 10 toabout 1100 mg/m². Lower dose regiments include doses of 10-200, 10-100,10-50 and 20-200 mg/m². Higher dose regimens include 200-400, 250-500,400-600, 500-800 600-1000 and 800-1100 mg/m². In one embodiment, thedose regimens range from 200-400 mg/m². In another embodiment, the doseregimens range from 250-500 mg/m². In yet another embodiment, the doseregimens range from 600-1000 mg/m². In some embodiments the agent isadministered daily, once per week, once every other week, or once permonth. In one embodiment, a dose regimen ranging from 200-400 mg/m² isadministered once a week. In another embodiment, a dose regimen rangingfrom 250-500 mg/m² is administered once every other week.

The doses may be constant over the entire treatment period, or they mayincrease or decrease during the course of the treatment. In oneembodiment, the agent is administered once a week and starts with theadministration of 200 mg/m² and increases to 300 mg/m² and 400 mg/m² inthe second and third weeks, respectively. In another embodiment, theagent is administered once every other week and is kept constant for theentire duration of treatment with the administration of 250 mg/m². Thedoses of the agent may be administered for at least one week, at leasttwo weeks, at least three weeks, at least four weeks, at least 6 weeks,or even at least 8 weeks. Adjusting the dose of the agent within theseranges for a particular subject is well within the skill of the ordinaryclinician.

The agent may be administered via any conventional route normally usedto administer a medicament including, but not limited to, intravenousroutes, parenteral routes (e.g., intradermal, intramuscular orsubcutaneous routes), oral routes and nasal routes. The agent may beadministered as a pharmaceutical composition in a variety of formsincluding, but not limited to, liquid, powder, suspensions, tablets,pills, capsules, sprays and aerosols. The pharmaceutical compositionsmay include various pharmaceutically acceptable additives including, butnot limited to, carriers, excipients, binders, stabilizers,antimicrobial agents, antioxidants, diluents and/or supports. Examplesof suitable excipients and carriers are described, for example, in“Remington's Pharmaceutical Sciences,” Mack Pub. Co., New Jersey (1991).In some embodiments, the agent may be administered via an IV infusion inan aqueous sugar solution. The agent may also be associated with anothersubstance that facilitates agent delivery. For example, the agent may beassociated into liposomes. The liposomes, in turn, may be conjugatedwith targeting substance(s), such as IgGFc receptors.

Exemplary embodiments of the present methods are provided in thefollowing examples. The following examples are presented to illustratethe methods of the invention and to assist one of ordinary skill inusing the same. The examples are not intended in any way to otherwiselimit the scope of the invention.

EXAMPLES Example 1 Microarray Analysis of Gene Expression ChangesInduced by MGBG

Blood samples were obtained from HIV infected patients (N=9) withpositive viral load and reduced CD4 counts, and non-HIV infected,healthy controls (N=6). Blood was collected into heparin anticoagulanttubes and mononuclear cells were isolated by Percoll gradientcentrifugation. The cells from each sample were split into 2: one-halfreceives MGBG at concentration of 10 μM and the other half received notreatment. All samples were then cultured overnight in RPMI media with10% fetal bovine serum at 37° C. under non-adherent conditions. The nextday cells were collected by centrifugation, washed one time with PBS,and lysed by resuspension in TRIZOL, and total RNA was preparedaccording to manufacturer's instructions (Invitrogen, Carlsbad, Calif.).The quality of the RNA was checked by visualizing rRNA bands using a2100 Bioanalyzer (Agilent Technologies, see Table 2 for ratio of 28 to18S rRNA). The RNA was then amplified for microarray analysis using a 2cycle amplification protocol according to the manufacturer'sinstructions (Affymetrix, Santa Clara, Calif.). The amplified RNAs werethen hybridized to Human Genome U133 plus 2.0 microarrays (Affymetrix)and expression data was obtained using an Affymetrix scanner and GCOSprotocols. Microarray data was analyzed with GeneSpring MicroarraySuite. Results were normalized using a set of 100 probe sets determinedby the manufacturer to have relatively invariant expression across mostcell types. The clinical parameters of the samples employed, the RNAquality analysis (ratio of 28 to 18S rRNA peaks) and the number of the54,675 probes called present are presented in Table 2.

TABLE 2 Clinical and RNA sample characteristics % CD14 CD4/ Viral %Reduction rRNA % Present Sample Type CD14/16 HLA-DR μl Load 14/16 14 − +− + H5-1293 HIV 68% 1390 1.5 1.7 46.5% 46.7% H5-1294 HIV 62% 935 12 46.41.0 1.9 40.0% 46.8% H5-1327 HIV 35% 805 43 712 <0% <0% 1.3 1.3 42.9%45.3% H5-1333 HIV 53% 861 567 13.6 71% 58% 1.7 1.6 46.6% 47.2% H6-004HIV 55% 1161 567 13.6 26% 26% 0.1 0.1 41.2% 43.5% H6-017 HIV 38% 882 24031.0 21% 0.1 0.2 44.2% 43.6% H6-071 HIV 54% 615 11 >500 1.5 1.4 42.5%41.4% H6-144 HIV 38% 557 54 43.0 53% 53% 1.2 1.0 40.3% 37.3% H6-145 HIV18% 438 567 13.0 91% 76% 1.3 1.5 46.5% 47.8% Average 46.8%   849 258124.7 60.3%   46.8%   1.1 1.2 43.4% 44.4% C6-019 Healthy 469 1.4 1.243.3% 41.6% C6-139 Healthy 41% 1105 54% 26% 1.8 1.8 48.0% 46.6% C6-146Healthy 73% 1950 61% 31% 1.7 1.5 47.5% 48.2% C6-186 Healthy 30% 634 1.81.8 49.6% 47.9% C6-187 Healthy 19% 667 1.8 1.7 48.8% 45.7% M6-177 ALS23% 496 1.7 1.8 47.2% 45.0% Average 37.2%   887 57.5%   28.5%   1.7 1.647.4% 45.8% Blank cells indicate no data was available. Viral loadvalues are in thousands per milliliter of plasma. % reduction indicatesdecrease in yield of CD14+ or CD14/16++ cells in the presence of 10 μMMGBG as determined on the 5th day of culture. Number of cells inpresence and absence of drug was quantitated by flow cytometry. rRNAindicates the ratio of the 28 to 18S rRNA peak as determined by the 2100Bioanalyzer from each sample in the in the absence (−) and presence ofMGBG (+). Note 1.8 to 2.0 is considered a perfect ratio. % Presentindicates number of probes called present by GCOS from each sample inthe absence (−) and presence of MGBG (+)

Using the average signals obtained from the MGBG and untreated samples,489 probes are down-regulated at least 1.5 fold and 382 probes areup-regulated at least 1.5 fold. Not all of these probes wereconsistently changed (e.g. always up regulated or down regulated). Forexample results obtained with a Probe set specific to TNFα are presentedin Table 2. Although the average change in the presence of MGBG for theTNFα Probe set was 8.7 fold up-regulated, inspection of the results ofindividual samples shows that 2 out of 6 controls and 6 out of 9 HIVinfected individuals were less than 2 fold changed in the presence ofMGBG relative to incubation without drug (Tables 3A and 3B). Similarly,although the average change in IL10 signal in the presence of MGBG was5.6 fold upregulated, inspection of the individual samples revealed thatincreased signals in the presence of MGBG primarily occurred in HIVinfected samples but not controls (Tables 4A and 4B).

TABLE 3A Results obtained with probe for TNFα. Probe ID = 207113_s_at

samples not significantly changed in the presence of MGBG are shaded

TABLE 3B Results obtained with probe for TNFα. Probe ID = 207113_s_at

samples not significantly changed in the presence of MGBG are shaded

TABLE 4A Results obtained with probe for IL10, Probe ID = 207433_at

samples not significantly changed in the presence of MGBG are shaded

TABLE 4B Results obtained with probe for IL10, Probe ID = 207433_at

samples not significantly changed in the presence of MGBG are shaded

Of all the genes that had changes in their average signals in thepresence of MGBG, six genes were selected for further analysis. Four ofthe genes were downregulated in the presence of MGBG and these includedFc gamma receptor I A (FCGR1a or the high affinity IgG receptor orCD64), leukocyte immunoglobulin-like receptor, subfamily A (with TMdomain), member 1 (LILRA1 also designated LIR6 or CD85 I), osteopontin(or secreted phosphoprotein 1 or SPP1), and secretogranin V or secretorygranule neuroendocrine protein 1 (SGNE1). Two of the proteins hadincreased signals in the presence of MGBG and these included adenosinedeaminase (ADA) and interleukin 24 (IL24). The signals obtained in themicroarray experiments with these six genes are presented in Table 5.The calculated fold change obtained in each sample is presented in Table6. FCGR1A and LILRA1 are down-regulated more than 2 fold by MGBG in 6 of9 and 5 of 9 HIV samples, respectively, but not in control samples whichhad much more variable changes than HIV infected individuals. SPP1 andSGNE1 exhibit 2 fold or greater reductions in signals in 12 and 13 ofthe 15 samples evaluated. Also note the lack of contrary changes. ADA isconsistently up-regulated by MGBG in all samples tested. IL24 isupregulated after MGBG exposure in HIV infected samples but notcontrols. Accordingly these six genes were further evaluated byquantitative real-time reverse-transcriptase PCR.

TABLE 5 Signals obtained with 6 most consistently changed genes FCGR1aLILRA1 SPP1 SGNE1 ADA IL24 Sample + − + − + − + − + − + − H5-1293 2642416 21 137 64 3875 14 45 1845 578 350 65 H5-1294 33 142 1 18 22 9523 8488 4290 84 1364 247 H5-1327 14 38 15 37 1684 2276 79 188 832 231 59 23H5-1333 347 874 164 81 487 2789 44 130 5472 376 577 127 H6-004 415 91740 44 1480 8780 17 751 769 130 111 25 H6-017 461 201 129 369 113 7689 71039 6724 3365 85 3376 H6-071 59 723 10 148 34 1938 10 99 6874 376 194010 H6-144 800 1012 65 413 143 247 13 40 1964 839 178 294 H6-145 15612078 246 187 458 2772 12 80 7488 336 375 14 Average 439 933 77 159 4984432 23 318 4029 702 560 465 C6-019 136 40 73 1055 53 5314 13 1603 53841509 578 2035 C6-139 51 91 42 313 81 1972 2 265 2900 1014 206 173 C6-146612 188 99 191 264 988 3 33 5895 1055 1549 474 C6-186 53 46 94 344 2321629 45 208 5781 1559 925 788 C6-187 1362 68 212 647 1519 3456 13 1226428 1519 163 748 M6-177 1435 494 277 3219 1647 1080 2 112 6731 2104 166360 Average 608 155 133 962 633 2407 13 391 5520 1460 598 763

TABLE 6 Fold changes of six most consistently changed genes.

Fold change (downregulation in upper panel; upregulation in lower group)in presence of 10 μM MGBG. Shaded cells were not significantly changed.Bold numbers indicate samples that had changes in signal of greater than2 fold in the opposite direction of the majority of samples for a givengene.

Example 2 Quantitative RT-PCR Confirmation of Genes Affected by MGBG

To evaluate the effect of MGBG on gene expression, blood samples wereobtained from patients (N=18, 10 HIV-infected and 8 Healthy Controls).Mononuclear cells were isolated via Percoll gradient centrifugation. Thecells were split into two tubes, one treated with 10 μM of MGBG and theother non-treated. The cells were then cultured overnight in RPMI mediaplus 10% fetal bovine serum at 37 C under non-adherent conditions,collected, lysed using Trizol solution, and total RNA prepared accordingto manufacturer's instructions (Invitrogen, Carlsbad, Calif.).

Approximately 200 ng of total RNA prepared from each sample wasconverted to cDNA using the First Strand cDNA Synthesis Kit for RT-PCR[AMV] kit (Roche Applied Diagnostics, Indianapolis, Ind.) according tomanufacturer's instructions. Around 5 ng of cDNA sample was then usedfor PCR performed on a LightCycler (Roche Applied Diagnostics,Indianapolis, Ind.) using the LightCycler FastStart DNA Master SYBRGreen I kit. PCR conditions are as follows: 1 cycle of denaturation at95° C. for 10 minutes, followed by 45 cycles of 95° C. for 10 seconds,68° C. for 10 seconds, and 72° C. for 16 seconds.

The sequences of gene-specific primers evaluated are listed below. Allsamples were also amplified with the human β-actin LightCycler-Primerset (Roche Diagnostics).

TABLE 7 Primers employed for Quantitative RT-PCR (QRT-PCR) GeneForward (5′ --- >) Reverse (5′ --- >) G1P3 AGCAGCGTCGTCATAGGTAATACAGGAGGATCACTTGAGGCT SEQ ID NO: 1 SEQ ID NO: 2 TGFB1CCAGCATCTGCAAAGCTCC TTGTACAGGGCCAGGACCTT SEQ ID NO: 3 SEQ ID NO: 4 CSF3RTGTTCGGCCTCCTGCTGTT GCTTCTTTTCATCCTCCTCCA SEQ ID NO: 5 SEQ ID NO: 6GPR43 TGCTACGAGAACTTCACCGAT GAAGCACACCAGGAAATTGA SEQ ID NO: 7SEQ ID NO: 8 MX2 TCTTCCCTGACCTTCACGAA CATTTATTAAGCAGTTACAATGCTGSEQ ID NO: 9 SEQ ID NO: 10 TNFSF10 CTACCTCATATCAGTTTGCTAGCAGCGATCTTTTAGTGGTGCCTCTT SEQ ID NO: 11 SEQ ID NO: 12 IFIT2CAGTAAAGAGCTTACTCCTGTAGCG AAGCCTCAGAATCTGCTCCATT SEQ ID NO: 13SEQ ID NO: 14 FCAR GACTTTCCCATGCCTTTCAT ACCGGAATCTGTAGTGCCCTASEQ ID NO: 15 SEQ ID NO: 16 ORM1 CGACAGGACCAGTGCATCTATAACTCTCCCAGTTGCTCCTT SEQ ID NO: 17 SEQ ID NO: 18 TNFRSF10cCCCAGCTGCTGAAGAGACAAT ATGATCCCTACGATGGTGCAT SEQ ID NO: 19 SEQ ID NO: 20CLECSF7 GGATAGCTGTTGTTTCAGAGAAAGG CTTCTCACAAATACTATATGAGGGCSEQ ID NO: 21 SEQ ID NO: 22 GPR86 AACACGACCATCGTAGGGTGATTTGAGGTGATGGTGGGATA SEQ ID NO: 23 SEQ ID NO: 24 CSPG2CACCGATGGCCATGTAAATA TGTCCAGGAAAAGCCATCTT SEQ ID NO: 25 SEQ ID NO: 26CD14 TGGAACAGGTGCCTAAAGGA ACAGGGTCGAACGTGCACA SEQ ID NO: 27SEQ ID NO: 28 NBS1 TGAATGAGGAGTTCTGGTACCT AGTCAAGCCACAGACTAGGTGTAASEQ ID NO: 29 SEQ ID NO: 30 CHI3L1 ACACAGATTTGAGCTCAGCCCATGTTTGGCTCCTTGGTGAT SEQ ID NO: 31 SEQ ID NO: 32 CLEC4EAGGTATTAAGCCCAGTGCCTAA GAAAATTATGTCTTTTGTGGGAACA SEQ ID NO: 33SEQ ID NO: 34 SIGLEC5 TTTCTGAGATGAAGTCGAGGG TCTGCTTGGAGCACTTAAACASEQ ID NO: 35 SEQ ID NO: 36 GPR109B AATTGTGTTGCTCCTGGAGGACAATGCCATTTCCTTTCCCA SEQ ID NO: 37 SEQ ID NO: 38 TNFa Quantitect cat #QT01079561 104 by amplicon 1Qgag TTGCCCATGGTTTCCAGAACAAGGGGATTTTTTCCTTGTGTTTTCA SEQ ID NO: 39 SEQ ID NO: 40 8pGAGAGTATGGATCTCAGGCGGT CATCGGTTGTAACATTACC SEQ ID NO: 41 SEQ ID NO: 42 PI3ATGGCCTTAGCTCTTAGCCAA GCTCTTGCGCTTTGACTTTA SEQ ID NO: 43 SEQ ID NO: 44IL1RN AGACCTTCTATCTGAGGAACAACCA TTGTCCTGCTTTCTGTTCTCG SEQ ID NO: 45SEQ ID NO: 46 IL6 TCCACTGGGCACAGAACTTAT TCTGGCTCTGAAACAAAGGASEQ ID NO: 47 SEQ ID NO: 48 IL1A TGCCTTCTGCTTTTAAGTTGCGATGAAGGGGTTCCCATAAA SEQ ID NO: 49 SEQ ID NO: 50 SPP1AGCCACAAGCAGTCCAGATTAT TTGACCTCAGAAGATGCACTATC SEQ ID NO: 51SEQ ID NO: 52 FCGR1A ACTCTGGGTTATACTGGTGCGA CCAAAGAGATTTCTAAATCCCACSEQ ID NO: 53 SEQ ID NO: 54 SGNE1 TCCCTGTGAATGACAGCATGTAAACTGCAAGAAATCTGAGCC SEQ ID NO: 55 SEQ ID NO: 56 LILRA1CAGTCAGGCAGAAGTATGCAAA TCCCTTTGTCCTAGAAAGTTGAGG SEQ ID NO: 57SEQ ID NO: 58 ADA GCTACCACACCCTGGAAGA CCGTTTGGTCATCTGGTAATCSEQ ID NO: 59 SEQ ID NO: 60 IL24 AACAGAGAGGGATGCTTGGATCACCAAGGGAAAGGGATGAT SEQ ID NO: 61 SEQ ID NO: 62

Of the genes evaluated, MGBG has the biggest effect on SPP1 (Table 8).It consistently down-regulates SPP1 levels in all of the unsortedsamples looked at, with fold changes between the untreated and thetreated of up to 300 in certain samples. IL1RN seems to bedown-regulated, though not as consistently (15 out of 18 samples) asSPP1. SGNE3 also seems to be mostly down-regulated, although the levelof inhibition in most samples is low. Genes that are up-regulated byMGBG include ADA and IL24. ADA is strongly induced and is upregulated inall but one sample. IL24 did not exhibit a consistent pattern of changein the presence of MGBG (Table 8). These results are in contrast to theresults obtained by QRT-PCR with many other genes, of which results with6 genes whose transcription appears unaffected by MGBG are presented inTable 9. Thus the effects of MGBG on osteopontin and adenosine deaminaseare highly specific.

TABLE 8 Fold change of 5 genes Identified by microarray by QRT-PCRDown-regulated Genes Up-regulated Genes Sample SPP1 IL1RN SGNE3 IL24 ADAH1293 −18.90 −4.92 1.79 2.50 3.01 H1294 −300.25 −2.31 −7.11 −2.41 3.76H1327 −2.14 −8.28 −2.50 −1.48 1.16 H1333 −18.90 −17.75 −4.41 −1.30 7.16H6_4 −20.11 −10.41 1.58 5.35 16.80 H6_17 −83.87 −3.73 −3.25 1.20 4.69H6_71 −6.59 1.12 −1.30 1.09 18.77 H6_144 −1.30 −1.73 −2.39 −1.13 2.33H6_145 −8.82 −1.36 −2.43 1.12 13.00 H1334 −95.67 −14.22 11.88 3.34C6_139 −7.89 −1.87 −1.48 2.57 5.90 C6_146 −1.95 −1.78 1.61 2.55 10.06C6_19 −46.21 8.63 −3.61 2.25 7.52 C6_186 −5.90 −2.27 −1.66 1.01 4.23C6_187 −2.11 1.59 −1.13 −1.17 7.21 C5_75 −6.15 −8.17 1.92 1.38 C5_76−1.28 −19.56 1.32 −0.73 C5_78 −467.88 −1.77 1.79 6.68 AVERAGE −60.9 −4.9−1.9 1.6 6.5 STDEV +/−124.2 +/−7.1 +/−2.5 +/−3.2 +/−5.3 MIN −467.9 −19.6−7.1 −2.4 −0.7 MAX −1.3 8.6 1.8 11.9 18.8 % >2 fold 83% 55% 50% 33% 83%Changed Table 8 shows the genes affected by MGBG in the unsortedsamples. The numbers represent the fold change between the untreated andthe treated. Negative numbers indicate fold reduction and the positivenumbers represent fold increase changes. Samples that were not evaluatedwith a particular gene are shaded grey. Significant reductions orincreases (>=2 fold) are in bold font. As shown, OPN or SPP1 is highlyinhibited after treatment with MGBG.

TABLE 9 Results obtained with six genes that were not affected by MGBGSample G1P3 MX2 IL6 NBS1 FCGR1A LILRA1 H1293 −2.85 1.13 1.74 −4.35−10.78 1.34 H1294 1.47 −2.11 −6.15 −8.51 −17.15 −4.79 H1327 1.22 −2.85−1.09 −2.51 −2.22 −1.31 H1333 −1.66 1.23 −5.46 −1.89 −3.86 1.28 H6_41.68 6.06 14.72 3.63 1.05 13.45 H6_17 1.92 2.03 −5.31 1.68 −1.61 −1.91H6_71 −1.25 −1.09 3.71 1.25 −1.84 1.01 H6_144 1.31 1.16 1.04 1.35 −1.01−3.10 H6_145 −1.25 −1.01 1.01 −1.56 −1.72 1.31 H1334 −2.03 4.35 −2.932.07 −3.73 C6_139 −1.14 1.56 −4.17 3.51 1.72 −2.87 C6_146 −2.04 −2.812.20 −1.09 5.94 1.52 C6_19 2.13 3.07 2.83 3.51 2.39 −3.97 C6_186 −1.141.16 −2.71 1.04 −1.97 −2.27 C6_187 1.22 −1.33 −4.06 4.08 19.03 1.11C5_75 −2.99 −1.77 1.74 2.31 1.16 C5_76 −6.63 −3.27 1.30 1.14 −0.41 C5_78−1.95 1.36 1.46 1.27 −0.43 AVERAGE 0.96 1.55 1.91 1.65 2.25 1.71 STDEV0.57 1.52 3.35 1.26 4.42 3.41 MIN −6.63 −3.27 −6.15 −8.51 −17.15 −4.79MAX 2.13 6.06 14.72 4.08 19.03 13.45

Example 3 Evaluation of Changes Induced by MGBG for OPN, ADA and OtherGenes

The changes induced by MGBG in different blood cell types weredetermined for osteopontin and adenosine deaminase and 7 other genes.For this experiment, blood samples were obtained from 6 healthy controls(3 men/3 women from 20 to 55 years of age) and mononuclear cells wereisolated via Percoll gradient centrifugation. The cells were thenincubated for 3 hours in RPMI media plus 10% fetal bovine serum at 37 C.At that point MGBG was added to 10 μM to half of each sample and thenthe cells were incubated at 37 C for 20 more hours. The cells were thenbound to magnetic beads coated with antibodies to human CD16. Boundcells (CD16 expressing) were separated from unbound cells using anAutoMACs according to manufacturer's instructions (Miltenyi, AuburnCalif.). Then CD16+ cells are released from the magnetic beads and boththe CD 16+ and CD 16− cells are combined with magnetic beads coated withantibodies to human CD 14. Antibody bound and unbound cells were thenseparated using the AutoMACs machine. This results in four differentcell fractions:

1. Cells that neither express CD16 nor CD14 (the −/− Cells). This ismostly T lymphocytes;

2. Cells that only express CD16. This includes contaminatinggranulocytes not removed by Percoll centrifugation and CD16+ monocyteswith very low CD14 expression;

3. Cells that express both CD16 and CD14. This includes monocytes thatare expressing CD16+ and macrophages (which express high levels ofboth); and

4. Cells that only express CD14. This would include most normal humanmonocytes.

After separation, cells of each fraction are washed with PBS and thenumber of viable cells determined by trypan blue exclusion. RNA is thenprepared from each cell group using TRIZOL, according to manufacturer'sinstructions.

The results obtained are presented in FIG. 2. It can be appreciated thatof the four cell fractions, the −/− cells are the most numerous with amedian yield of 4.2 million cells. Approximately ten fold less CD14+ anddouble positive cells were isolated (4.7 and 3.8×10⁵ cells,respectively). The lowest number of cells is in the CD16 single positivefraction which had a median yield of 6.0×10⁴ cells. β-actin levels asmeasured by QRT-PCR accurately reflected cell counts with the highestthreshold cycles obtained from the CD16+ cells and the lowest valuesfrom the double-negative cells. For osteopontin, the highestβ-actin-normalized signals were obtained from CD 14+ monocytes andCD14/16++ monocyte and macrophages. Upon exposure to osteopontin RNAlevels in the CD14+ monocyte fraction decreased the most with a medianreduction of 4.8 fold (0.21 of no MGBG) which was significantly lessthan 1.0 (no change) by the Wilcoxon signed rank test (p=0.03). Incontrast changes induced by MGBG in CD14/16++ cells, CD16+ cells, anddouble negative cells were 0.61, 1.12, and 0.70, respectively. None ofthese changes were significantly different than 1.0. Thus, in healthyindividuals MGBG exposure reproducibly reduces osteopontin RNA signalsin monocytes.

For adenosine deaminase, levels of RNA signal were approximately equalin all four cell fractions (from 1 to 10% of actin levels). Exposure toMGBG resulted in significant increases in ADA transcription in CD14+monocytes (median change 10.8 fold, p=0.0156). Much lower but stillsignificant changes were seen in CD14/CD16++ cells (median change 2.8fold). Double negative cells and CD16+ cells did not changesignificantly. Thus as is the case for osteopontin, RNA changes areprimarily seen in monocytes after exposure to MGBG. Smaller changes arealso detected in CD14/CD16 double positive cells. Thus monocytes andmacrophages are the primary cellular targets of MGBG.

To determine if MGBG treatment might be globally affecting monocytes andmacrophages we looked at four additional gene that are known to behighly expressed in monocytes and macrophages. The genes were CD14(which also provides a level of quality control on our cell separations)and interleukin 1 receptor antagonist (IL1RN), G coupled receptor 43(GPR43, also known as the free fatty acid receptor), and the cytokineTNFα. For all four genes levels of RNA in monocytes and macrophagesranged between 10 to 100 times greater than that seen in double negativelymphocytes. IL1RN CD14, and TNFα had approximately equal levels of RNAsignal in both CD 14+ monocytes and CD14/CD16++ macrophages. For GPR43,median RNA levels were 5 to 10 fold higher in CD14/16++ cells relativeto CD14+ monocytes. Exposure to MGBG resulted in no clear trend inexpression changes. In particular exposure to MGBG significantly loweredRNA signal of TNFα in monocytes (median signal=0.32, p=0.03) but not thesignals of the other 3 genes. In contrast exposure to MGBG caused aslight increase in GPR43 expression (media signal=2.4, p=0.03) and aslight decrease in RNA signals for CD14 (median signal=0.55, p=0.02) inCD14/CD16++ macrophages. Thus evaluation of other monocyte andmacrophage specific genes did provide evidence for a global andsystematic effect of MGBG on gene expression in those cells. Thus themore profound effects of MGBG on osteopontin and adenosine deaminase arespecific.

Example 4 Effect of MGBG on Osteopontin Protein Secretion

Peripheral Blood Mononuclear Cells (PBMCs) were isolated from patientsdiagnosed with Amyotrophic Lateral Sclerosis (ALS), Alzheimer's Disease(AD), Human Immunodeficiency Virus (HIV), Breast Cancer, AIDS Dementia,and healthy donors. The heparinised blood was mixed with equal volumesof sterile phosphate-buffered saline (Ca⁺⁺, Mg⁺⁺ free PBS) and layeredover Percoll (Amersham Biosciences, Piscataway, N.J.) at 1.087 g/ml. Thecells were centrifuged and the mononuclear cell layer was collected. ThePBMCs were cultured in a concentration of 1×10⁶ cells/ml in non-adherentconditions in polypropylene tubes containing complete media (RPMI 1640,10% fetal bovine serum (HyClone, Logan, Utah), 1% Sodium Pyruvate) andincubated in a 5% CO₂, 37° C. humidified incubator. The cells were grownin the presence of 0 μM, 0.1 μM, 1 μM, 10 μM, or 100 μM MGBG pluscomplete media and the cell culture supernatants (CCS) were isolated onthe fifth day and frozen at −20° C. until ready to be tested. HumanOsteopontin (OPN) levels were measured with the Human Osteopontin ELISAkit (R&D Systems, Inc., Minneapolis, Minn.) according to manufacturer'sinstructions. The CCS were tested in duplicate and the Magellan v. 4.0software (Salzburg, Austria) was used to analyze the results.

The average OPN levels (ng/ml) in 0 μM, 0.1 μM, 1 μM, 10 μM, and 100 μMMGBG were measured. The results are depicted in FIGS. 3-7 as a graphcomparing diseased sample with normal sample. In FIGS. 3-7, each pointon the graph is the average of each sample with standard errorrepresented as error bars. The solid lines represent diseased cells andthe dashed lines represent normal cells.

The average OPN levels obtained from the CCS of healthy/normal samples(n=3) were 270, 9.69, 4.85, 1.07, and 0.14 ng/ml in cultures with 0 μM,0.1 μM, 1 μM, 10 μM, and 100 μM MGBG, respectively. The correspondingaverage OPN levels obtained from the CCS of breast cancer samples (n=3),as shown in FIG. 3, were 125.96, 20.89, 8.87, 2.12, and 0.84 ng/ml. Thecorresponding average OPN levels obtained from the CCS of the AD sample(n=1), as shown in FIG. 4, were 0.926, 0.477, 0.201, 0.136, and 0 ng/ml.For AD, each point on the graph is the average of the duplicates testedon the ELISA plate. The corresponding average OPN levels obtained fromthe CCS of HIV samples (n=2), as shown in FIG. 5, were 78.698, 11.833,3.481, 0.447, and 0 ng/ml.

PBMCs obtained from ALS and AIDS Dementia patients were tested with 0 μMand 10 μM only. The average ALS (n=2) CCS values, as shown in FIG. 6,were 525.644 (0 μM) and 17.669 (10 μM); and the average AIDS DementiaCCS (AIRL2005-374) value, as shown in FIG. 7, was 30.958 (0 μM) and0.8905 (10 μM). Cell viability data showed that the cells at each MGBGconcentration were at least 75% viable when the CCS were collected.

Additionally, cells being targeted by MGBG were investigated. Phenotypicanalysis showed that increasing concentrations of MGBG resulted indecreasing yields of CD 14+ macrophages but did not affect levels ofCD16+/CD14− cells. To elucidate the effects of MGBG and OPN on cellmotility, migration assays were conducted. MGBG at 10 μM inhibitedmacrophage Osteopontin production from >200 ng/mL to nondetectablelevels while simultaneously inhibiting HeLa cell invasion into aMatrigel matrix. The results show that MGBG inhibits OPN production ofmononuclear cells in vitro and that inhibition of OPN by MGBG issufficient to inhibit cell migration.

Example 5 Effect of Removing Extracellular Osteopontin on MacrophageDifferentiation

To observe the effects of OPN blocking antibody on macrophageactivation, 10 μg of antibody (Immuno-Biological Laboratories, Inc.,Minneapolis, Minn.) was added to non-adherent PBMC cultures for 24 and72 hours. The cells were labeled with mouse anti-human CD14-TriColor(Invitrogen, Carlsbad, Calif.) and mouse anti-human CD16-FITC(fluorescein isothiocyanate; Dako, Carpinteria, Calif.) according to themanufacturer's suggestion; an isotype control was also used. CD 14+monocytes and CD14+/CD16+ macrophages were analyzed by flow cytometry at24 and 72 hours after cell isolation (BD FACScan, San Jose, Calif. withFACS Express 3 software). Results showed that incubation withneutralizing osteopontin antibody increased the yield of CD14+ monocytes5.9 fold at 24 hours (2.5% CD14+ in presence of SPP1 Ab vs 0.4% withcontrol Ab) and 16.9 fold at 72 hours (3.7% CD14+ vs 0.2%).Additionally, at 24 hours the yield of CD14/16++ macrophages was reduced4.5 fold in the presence of neutralizing osteopontin antibodies (0.6% vs2.5% in presence of control Ab). Additionally CD14/16++ cells isolatedin the presence of osteopontin neutralizing antibodies never achievedthe typical increase in side scatter seen under normal conditions. Thusremoval of osteopontin prevents normal macrophage differentiation andincreases the prevalence of CD 16− monocytes.

Example 6 Effect of Adding Exogenous OPN on Macrophage Yield

To determine the role of exogenous recombinant human OPN (rOPN) inmacrophage activation, 10 μg/ml rOPN(R&D Systems, Inc., Minneapolis,Minn.) was added to non-adherent PBMC cultures grown in complete or inserum free medium for 24 and 72 hours, and analyzed via flow cytometry.The cells were labeled with α-axis) mouse anti-human CD 14-TriColor(Invitrogen, Carlsbad, Calif.) and (y-axis) mouse anti-human CD16-FITC(fluorescein isothiocyanate; Dako, Carpinteria, Calif.) according to themanufacturer's suggestion. As seen in FIG. 8, rOPN increases CD14+/CD16+cell population. At 24 hours, cells incubated with rOPN in completemedium had no significant accumulation of CD14+/CD16+ cells incomparison to control (no rOPN in the medium). However, at 72 hours a11-fold increase in CD14+/CD16+ cells were observed in cells incubatedwith rOPN in comparison to control. A 2.97-fold increase in CD14+/CD16+cells was also observed with cells cultured with rOPN for 72 hours inserum free conditions in comparison to serum free control. Shown in theupper right quadrant in FIG. 8 is the percentage of cells that aredouble positive for CD14 and CD16 surface markers.

Example 7 Osteopontin Induces Increased Macrophage Activation andCytokine Secretion

We investigated macrophage activation via flow cytometry and levels ofpro- and anti-inflammatory cytokines in cell culture supernatants ofmononuclear cells. 10 μg/ml recombinant human OPN (rOPN) was added tomononuclear cells cultured for 3 days, with and without fetal bovineserum (FBS) in cell culture medium, and analyzed via flow cytometry. At24 hours, cells incubated with rOPN and FBS had no significantaccumulation of CD14+/CD16+ cells in comparison to FBS-only control.However, at 72 hours, a 12-fold increase in CD14+/CD16+ cells wereobserved in cells incubated with rOPN in comparison to control. Anincrease in CD14+/CD16+ cells was also observed with cells cultured inserum free medium and rOPN in comparison to control. To observe theeffects of OPN neutralizing antibody on macrophage activation, 10 μg ofantibody was added to cultures for 3 days. The amount of CD14+/CD16+cells was inhibited by 3.97-fold at 24 hours and 3.76-fold at 72 hours.We also investigated the levels of pro- and anti-inflammatory cytokinesin all cell culture supernatants of each time point. On average, IL-6,IL-1β, TNF-α, IL-10, and IL-12p40 were at least 50-fold higher with rOPNat 24 and 72 hours in all culture conditions in comparison to control.IL-12p70 and IL-4 levels had no significant difference at any time pointand culture condition. In conclusion, we have found that rOPN increasedmacrophage activation by the third day. Heightened levels of most of thecytokines measured were also observed when the cells were exposed torOPN, suggesting that OPN induces mononuclear cells to secrete thesecytokines.

Example 8 Effect of MGBG on Osteopontin In Vivo

Many chronic diseases are associated with elevation in osteopontinproduction in diseased tissues. In order to test whether MGBG, whichregulates OPN production in vitro, would have the same effect in vivo,an animal model study was performed. Simian immunodeficiency virus (SIV)infected rhesus macaques develop AIDS and a macrophage infiltrativeprocess of the brain associated dementia within months after infection.MGBG was used in two trials in SIV-infected animals. Rhesus macaqueswere infected with SIVmac251 and depleted of CD8+ T lymphocytes byadministration of a humanized CD8-depleting antibody. This resulted in arapid depletion of CD8+ T lymphocytes and a very rapid viral infectionwith a short time course to AIDS. These monkeys were then treated withMGBG as described.

In the first trial three animals (2 treated and 1 untreated) were used.The animals that were treated with MGBG were initially given 200 mg/m²MGBG, and then treated 7 and 14 days later with 300 mg/m² and 400 mg/m²,respectively of MGBG. While the untreated animal developed SIVE by thethird week, the treated animals showed no indications of SIVE or DRGinfiltration. The trial was halted when the treated animals developedsevere GI toxicity at an MGBG dose of 400 mg/m².

In the second trial 2 treated and 2 untreated animals were used. In thistrial the animals were treated with a bi-weekly dosing of 250 mg/m²MGBG. The untreated animals were SIVE+ in 50 days post-infection anddeveloped AIDS/SIVE within 85 days. The treated animals showed noevidence for SIV disease. Further, no drug related toxicity was seenthrough 8 cycles of MGBG.

In both trials, the infected untreated animals developed AIDS anddementia, but none of the treated animals became ill. FIG. 9 shows thatin the infected, untreated animal brains, characteristic macrophageinfiltration was observed, but with MGBG (PA001) treatment, no pathologywas observed. SIV-infected macaques treated with 4 doses of MGBG (PA001)demonstrated absence of SIV-associated macrophages in the frontal cortexcompared to non-treated.

These same brains stained with an antibody to OPN, showed that treatmentwas associated with complete removal of OPN whereas the infecteduninfected animal's brain had large concentrations of OPN (FIG. 10).Similarly, as shown in FIG. 11, lymph nodes from the animals showeddifferential levels of OPN staining with treatment (B146 and CB 18)associated with markedly decreased levels of OPN. These results showthat MGBG treatment of an OPN associated disease turns off OPNproduction in vivo with associated reversal of pathologic processes.

Example 9 Effect of MGBG on Osteopontin RNA and Protein Production

Polyamine analogs SL47 and SL93 as well as MGBG were compared withdexamethasone and growth factor MCSF in an OPN production inhibitionassay. 10 μM of each drug was added to un-stimulated PBMC cultures attime=0 and cells were harvested 1,3, and 6 days later for quantitativeOPN RNA and protein production analysis. FIG. 12 shows the effect of(from left to right) SL47, SL93, MGBG, dexamethasone, and MCSF on OPNRNA As seen in FIG. 12, OPN RNA expression was inhibited in a timedependant manner for SL47, SL93, and MGBG. Dexamethasone and MCSF, onthe other hand, had no effect on OPN RNA expression.

Similar results were observed for OPN production. FIG. 13 shows thequantitative levels of OPN produced in cultures treated with SL47, SL93,MGBG, and dexamethasone as described above. Complete inhibition of OPNprotein production was observed in PBMCs treated with MGBG as well aspolyamine analogs SL47 and SL93.

All references and publications cited herein are incorporated byreference in their entirety.

It should be noted that there are alternative ways of implementing thepresent invention. Accordingly, the present embodiments are to beconsidered as illustrative and not restrictive, and the invention is notto be limited to the details given herein, but may be modified withinthe scope and equivalents of the appended claims.

1. A method of treating, or alleviating the symptoms of, a conditioncomprising administering to a subject in need of such treatment aneffective amount of MGBG or a salt thereof, wherein the condition isselected from the group consisting of inflammatory diseases, autoimmunediseases, Crohn's disease, Parkinson's disease, multiple sclerosis (MS),inflammatory bowel disorder, amyotrophic lateral sclerosis (ALS),hepatitis, HBV, HCV, nephritis, cerebritis, rheumatoid arthritis, type 2diabetes, glomerulonephritis, cardiac fibrosis and angiotensin type IIassociated hypertension, osteoporosis, a mast cell produced IgE mediatedhypersensitivity immune reaction, peripheral sensory neuropathyassociated with HIV infection or diabetes mellitus, asthma, autism,dermatomyositis, frailty, obesity, primary biliary cirrhosis, primarysclerosing cholangitis, post-radiation syndrome, psoriatic arthritis,sarcoidosis, scleroderma with or without pulmonary fibrosis, a kidneyrelated autoimmune condition, diabetic nephropathy, a diabetic vascularcomplication and a lymphoproliferation related autoimmune condition. 2.The method of claim 1, wherein the condition is an autoimmune conditionor disease.
 3. The method of claim 1, wherein the condition isinflammation.
 4. The method of claim 1, wherein the condition ispsoriatic arthritis, peripheral sensory neuropathy, multiple sclerosis,lupus, inflammatory bowel disorder, arthritis, nephritis, or cerebritis.5. A method of treating, or alleviating the symptoms of multiplesclerosis comprising administering to a subject in need of suchtreatment an effective amount of MGBG or a salt thereof.